1,4-bis-acylpiperazines

ABSTRACT

1,4-BIS-ACYLIPERAZINES IN WHICH THE ACYL GROUP IS MERCAPTOACETYL OR A-SUBSTITUTED MERCAPTOACETYL ARE POTENT MUCOLYTIC AGENTS WHICH ARE NOT ONLY EFFECTIVE TOPICALLY BUT ALSO SYSTEMICALLY ON ORAL OR PARENTERAL ADMINISTRATION TO ANIMALS.

May 7, 1914 AR TI Em 3,809,697

1,4-BIS-ACYLPIPERAZINES Filed Oct. 7, 1971 FIGURE 1. REDUCTION IN sPuTuM visCOslTy @Y N-ACE.T L.CY5TEINE(NAC) AND |,4- bi5 MERCAPTOACETYL PIPERAZKNE. (MAP) 2 i 2 Oman 2 202925 I a E- 7 I] e- Z 70 VlfBCOSITY OF OFZ16\NAL.

United States Patfl ABSTRACT OF THE DISCLOSURE 1,4-bis-acylpiperazines in which the acyl group is mercaptoacetyl or tit-substituted mercaptoacetyl are potent rnucolytic agents which are not only etfective topically but also systemically on oral or parenteral administration to animals.

FIELD OF THE INVENTION DESCRIPTION OF THE PRIOR ART The following publications constitute the closest prior art known to the inventors with respect to this invention.

J '(1) A. L. Sheffner, Ann. N.Y. Acad. Sci., 106, 298, 310

(1963), (2) E. R. Atkinson et al., J. Med. Chem., 8, 29-33 3 T. Irikura et al., ibid. 11, 801-804 (1968).

In reference No. 1 the testing of a variety of compounds for mucolytic activity in vitro was reported. In an attempt to a correlate mucolytic activity with chemical structure,

the author concluded that the free sulfhydryl group was necessary. -N-acetyl-L-cysteine was singled out for detailed study and this substance subsequently became commerically available under the trademark Mucomyst as a topically effective mucloytic agent. No piperazine derivatives were described.

While N-acetyl-L-cysteine has been a highly successful product and enjoys the leading market position for mucolytics, further improvement is nevetheless desirable with respect to potency and oral use. N-acetyl-L-cysteine is administered by inhalation, a form of topical treatment, .and

has not been recommended for oral use as a mucolytic.

In reference N0. 2 preparation of a variety of amides of thioglycolic acid was described and test results therewith as radioprotective agents summarized. No piperazine derivatives were described.

A series of 1,4-bis-acylpiperazines was described in reference No. 3. The only sulfur containing member of this series is 1,4-bis(phenylthioacetyl)piperazinewhich was disclosed to have analgetic activity. No free sulfhydrylcontaining compounds were described.

SUMMARY The present invention provides the previously unknown compounds shown in Formula I.

' R Formula 1 i In this formula, R and R may be'the same or different and are selected from the group consisting of hydrogen; and

3,809,697 Patented May 7, 1974 lower alkyl having 1 to 4 carbon atoms. R and R are preferably the same and in the most preferred embodiments they are hydrogen. X and Y are selected from the group consisting of hydrogen, alkanoyl having up to 20 carbon atoms, and aroyl having from 6 to 11 carbon atoms. The salts of those substances in which X and Y are hydrogen are also part of the present invention. The preferred salts are those prepared from pharmaceutically acceptable bases including the sodium, potassium, calcium,

barium, zinc, aluminum, magnesium, bismuth, and ammonium salts, and the salts of non-toxic organic bases suchlas the non-toxic primary, secondary, and tertiary aliphatic amines and quaternary ammonium bases e.g., trimethylamine, triethylamine, ethanolamine, methylethanolamine, triethanolamine, benzyltrimethylammonium hydroxide, etc. In other words, in addition to hydrogen, al-

kanoyl, and aroyl as described above, X and Y may also be cationic salt forming species including ammonium, quaternary ammonium, pharmacologically acceptable metal ions, and pharmacologically acceptable protonated amines. Pharmacologically acceptable as used to de scribe the metal, ammonium, and amine salts refers to those cationic species which do not contribute appreciably to the toxicity of the product nor to its pharmacological activity. In other words, the cationic components of the salts of Formula I are pharmacologically inert.

The symmetrical compounds of Formula I in which X andY are identical and R and R are identical are preferred because they are easier and cheaper to manufacture. They are shown in Formula II in which the symbols R and X have the same meaning as above xst jno ON NCOCHSX R Formula II The l,4-bis(mercaptoacyl)piperazines of Formula I are useful as mucolytic agents. Those in which X and Y are hydrogen andthe salts thereof are active in vitro, when applied topically, and systemically in animals. They are more potent than N-acetyl-L-cysteine. When administered orally, they have a prolonged duration of action as compared to N-acetyl-L-cysteine. The esters thereof, that is weight are used. Solutions in aqueous vehicles having concentrations of 0.5% to 5% by weight or dry powders are preferred for inhalation.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is made of 4 graphs plotted on rectangular cof ordinates on the same set of axes. The ordinate is calibrated in percentage units with reference to the viscosity of the test sputum sample at the beginning of the experimentQThe abscissa refers to time and is calibrated in bis-(mercaptoacetyl)piperazine.

DESCRIPTION OF SPECIFIC EMBODIMENTS The superior mucolytic activity of 1,4-bis(niercaptow period. This reduces the amount of sputum on the test plate to 1 ml. and reduces the viscosity of the specimen to a reproducible value which is necessary because of the thixotropic properties of sputum. The rotation rate is then reduced to give a convenient reading on the instrument, and a solution of the test drug having a volume of 0.2 ml. is then added to the sputum cup' and viscosity readings as a percentage of the original are recorded at time intervals of l min. for a period of Typical results are shown in FIG. 1. T

FIG. 1 is a collection of 4 graphs in which the viscosity reading taken from the viscometer and expressed as a percentage value with respect to the viscosity of the specimen just prior to contact with the mucolytic agent (zero time) is plotted versus time at 1 min. intervals for 15 min. One of the graphs refers to the mucolytic effect of 0.3 M N-acetylcysteine, referred to in the graph as NAC and the other three graphs to the mucolytic effect of solutions having various concentrations of 1,4-bis(mercaptoacetyl)piperazine, referred to in the figureas MAP. The figure shows that the solution of MAP having a concentration of 0.0094 M has substantially the same mucolytic effect as a solution of NAC having a concentration of 0.3 M. On a molar basis MAP is therefore approximately 30 times as active as NAC and on an equivalent weight basis with respect to the sulfhydryl group, approximately 15 times as active. I Y When rats are treated orally with a dose of 180 mg./kg. (0.77 millimole/kg.) of 1,4 bis (mercaptoacetyl)piperazine, plasma sulfhydryl concentration ,is elevated and remains so for at least 6 hrs., but returns to normal within 18 hrs. Similar results are obtained with a similar dose on a molar basis (245 mg./kg.) of 1,4-bis(acetylthioacetyl)piperaz'ine which indicates that the body contains enzymes which cleave the thioester to,the .thiol compound.'Lung sulfhydryl concentrations were also elevated following oral administration of either compound. Plasma sulfhydryl concentrations following oral administration of N-acetyl-L-cysteine (500 mg./kg., 3.07 millirnoles/kg.) are elevated briefly following oral administration but return to normal within 6 hours. I

Rats when exposed to an atmosphere containing ap- I proximately 450 p.p.m. of S0 on a chronic basis secrete abnormally high amounts of mucus into the bronchial tree. This is reflected in the amount of solids collected from lung washings of the rats. Plasma sulfhydryl concentrations are also depressed by this treatment, andv the cellular composition of the epithelium of the lung undergoes changes. These'elfects serve as parameters which may be used to measure mucolytic activity in vivo. 1

Two groups of 10 rats each were exposed to an atmosphere containing 450 p.p.m. of S0 for 3 hrs. a day, 6

days a week, until they had endured a total of 30' hrs. ,Of exposure. During this time one group was treated orally with 180 mg./kg. of body weight per day of 1,4- bis(mercaptoacetyl)piperaz'ine. A third group of 10 rats which "was neither exposed to S0 nor treated was maintained as a control group. At the end of the experiment the animals were sacrificed, tracheal mucus collected and weighed, the volume of lung mucus measured by centrifuging lung washings, and blood plasma sulfhydryl condrug treatment.

from S0 exposure were significantly alleviated by the Eflect of oral treatment with 1,4-bis(mercaptoacetyl)piperazine on rats breathing 450 p.p.m. of SO: in air-dose 180 mgxlkg.

1 Average. 1 I 1 Determined colorimetrically according to the method of J. Sedalak and R. S. Lindsay Anal. Biochemistry 25, 192 (1968) using N-acetylcysteine as sulfhydryl in the color standard.

1 'I'he compounds of Formula I are synthesized by the application of known methods for the preparation of mercaptans and thioesters. Useful intermediates for this purpose are the corresponding halogen compounds, namely the substances of Formula I wherein chlorine, bromine, ,or iodine are substituted for X and Y. The 1,4-bis (haloacyl)piperazines whichserve as intermediates are converted to the mercaptoacyl compound by reaction,jfor instance, with sodium hydrosulfide, sodium thiocarbonate, or potassium hydrosulfide. Alternatively, and preferably potassium thiolacetate may be caused to react with the 1,4-bis(haloacyl)piperazines by replacement of the balm gen atoms with the CH COS-group. It has already been stated that these thioesters are of interest as biologically active and products in themselves in addition to serving as intermediates for preparing the 1,4-bis(mercaptoacyl) =piperazines on hydrolysis.

The higher alkanoyl esters of Formula I may be prepared by esterification of the l,4-bis(mercaptoacyl)piperazines by reaction thereof with an acylating agent such as an :acid chloride or anhydride. Alternatively, the 1,4- .bis(haloacyl)piperazine intermediate may be caused to react with the sodium or potassium salt of the corresponding alkanoyl or aroyl thiolacid. The latter are prepared by reaction of H 5, with the corresponding alkanoyl or aroyl halide'or anhydride. I

The unsymmetrical compounds of Formula I, that is those substances where R and R are different from one another, or X and Y are different from one another, may be prepared by commencing with a piperazine having a readily removable blocking group on the nitrogen atom 1 in the 1-position such as the ethoxycarbonyl, formyl, or

in the 1-position is transformed in corresponding fashion with the desired acyl group.

The foregoing synthetic principles are illustrated by the I following preparative procedures.

' (1) 1,4 bis(chloroacetyl)piperazine. Chloroacetyl chloride," 153.6 grams (1.36 mol.) is added slowly with stirring toa mixture of 120 g. (0.62 mol.) of piperazine hexahydrate, and 136 'g. (1.62 mol.) of sodium bicar- 'bonate in '600 ml. of water. Cooling is used to maintain 60 1 the reaction temperature in the range 15-25 C. The product precipitates in the course of the reaction and after stirring the mixture for several hours at room temperatur'e, the product is collected by filtration, washed on the filter with water, and dried; yield 110 g. (74%), MP.

(2) l,4-bis(acetylthioacetyl)piperazine.--A mixture of 6g. (0.025 mol.) of 1,4-bis(chloroacetyl)piperazine, ml. of acetone, and 6.5 g. (0.0555 mol.) of potassium thiolacetate is warmed at 40-45 C. 2 hrs. The mixture is concentrated by distillation of the acetone and the residue is stirred with water-to yield 6.5 g. (82%) of the desired product, M.P. 143-1445 C. i

'-.-Analysis.Calcd. for C H N O S (percent): C,

45.26; H, 5.70;.N, 8.80. Found (percent): 0, 45.26; H,

(3) 1,4 bis(mercaptoacetyl)piperazirie; disodium salt.- A mixture of :4 g. of 1,4 bis(acetylthioacetyDpiperazine and250 ml. of ethanol is; stirredun'der an atmosphere' of nitrogen and 52 ml. (0.13 mol.) of 10% aqueous sodium hydroxide is added thereto. A clear solution" forms from which the desired disodium salt precipitates as a white solid. The mixture is stirred for 2 hrs. at room temperature and then the product is collected by filtration. It is washed on the filter first with 3:2. ethanol-water and then with 95% aq. ethanol.

(4) 1,4-bis(mercaptoacetyl)piperazine.-=The disodium salt of the preceding preparation is dissolved in 160 ml. of methanol under an atmosphere ofnitrogen and slowly treated with 14 ml. of 5 N ethanolic I-ICl. By-productsodium chloride is discarded after filtration, and the filtrate concentrated to one-halfl its volumelby distillation.- The product separates as a solid and is collected on a filter; yield 6 g. (75% M.P. 107.5-109" 'C. v

Analysis.--Calcd. for C H N O S (percent) C, 41.00; H, 6.02; N, 11.96. Found (percent): C, 40.74; H, 5.99; N, 12.10.

(5) 1,4 bis(2 chloropropionyl)piperazine.Except for the use of an equimolar amount 2-chloropropionyl chloride in place of chloroacetyl chloride, the procedure for the preparation of this compound is the same as that used in Procedure 1; 56% yield. Recrystallization by dissolving in hot acetonitril'e followed by dilution with isopropyl ether gives the pure product, M.P. 162.5-164 C.

(6) 1,4 bis(2 acetylthiopropionyl)piperazine. A mixture of 10 g. (0.0375 mol.) of 1,4-bis(2-chloropropionyl)piperazine, 9.2 g. (0.08 mol.) of potassium thiolacetate and 200 ml. of acetone is heated overnight under reflux. The solvent is evaporated under reduced pressure. The residue is stirred with a mixture of chloroform and water. The organic layer is separated, washed with water, dried over anhydrous potassium carbonate, and concentrated to an oil. On solution of this oil in ethyl acetatelight petroleum ether, the product crystallized; yield, 7.7 g. (60%). Recrystallization from ethanol gives the purified compound; M.P. l3l.5133.5 C.

Analysis.Calcd. for C H N O S (percent): C, 48.53; H, 6.40; N, 8.09. Found (percent): C, 48.77; H, 6.64; N, 8.12.

(7) 1,4 bis(2 mercaptopropionyl)piperazine.-To a mixture of 5 g. (0.0144 mol.) of 1,4-bis(2-acetylthiopropionyl)piperazine and 75 ml. of anhydrous ethanol stirred under an atmosphere of nitrogen there is added 32 ml. of 10% aqueous sodium hydroxide. The reaction mixture is stirred for 4 hrs. and then concentrated to an oil which is slurried with methanol and acidified with ethanolic hydrogen chloride. The resulting solid is collected and washed with methanol to give the crude product. It is purified by slurrying with water, filtering and washing first with water and then with acetone; yield, 3 g. (79%). Further purification is achieved by recrystallization from anhydrous ethanol; M.P. 17l172.5 C.

Analysis.Calcd. for c,,,H,,N,o,s, (percent): C, 45.77; H, 6.91; N, 10.68. Found (percent): C, 45.90; H, 6.76; N, 10.62.

(8) 1,4 bis(propionylthioacetyl)piperazine. To a mixture of 15 g. (0.0639 mol.) of 1,4 bis(mercapto acetyl)piperazine and 250 ml. of chloroform under an atmosphere of of nitrogen there is added slowly with stirring 57 ml. of 10% aqueous sodium hydroxide and then after cooling to 0-5 C. 12 g. (0.129 mol.) of propionyl chloride during 10 min. After stirring overnight at room temperature, the organic layer is separated, washed with water, dried over anhydrous magnesium sulfate and concentrated to yield the solid product. The compound is purified by slurrying with warm ethyl acetate, filtering insoluble impurity, diluting the filtrate with light petroleum ether, and cooling; yield, 13.5 g. (61%). Further recrystallization from the same solvent combination yields the pure compound, M.P. 94-96 C.

Analysis- Calm. for C H N O SQ (percent): C,

48.53; H, 6.40; N, 8.09. Found (percent): 0., 48.20; H, 6.-25;-N,-8'.32.

(9) 4 chloroacetyl 1 formylpiperazine.-1-formylpiperazine is chloroacetylated in the manner described in Procedure 1 to provide this intermediate.

(10) 4 acetylthioacetyl l formylpiperazine.-The reaction between 4 chloroacetyl 1 formylpiperazine and potassium thiolacetate is performed according to Procedure 2 to provide this intermediate.

v(11) 1 Q mercaptoacetylpiperazine.-Selective removal of the blocking formyl group from 4-acetylthioacetyl-lformylpiperazine is accomplished by tretment with sodium hydride in dimethoxyethane. Hydrolysis of the thioester group also occurs under these conditions.

12) l mercaptoacetyl 4 (2 mercaptopropionyl piperazine.- l mercaptoacetylpiperazine is converted to 1 mercaptoacetyl 4 (2 chloropropionyl)piperazine by reaction thereof with 2-chloropropionyl chloride according to Procedure 1 modified by substitution of these reactants. ,-'Ihe v resulting l-mercaptoacetyl4(2-chloropropionyl)piperazine is then converted to the desired product by application of the methods of Procedures 6 and 7 according to which the chloropropionyl group is successively transformed to acetylthiopropionyl and then to mercaptopropionyl.

(13) 1,4 bis(palmitoylthioacetyl)piperazine. An equimolar amount of palmitoyl chloride is substituted for the propionyl chloride specified in Procedure 8 and the procedure is then repeated as described to provide the desired bis-palmitoyl ester.

14) 1,4 bis(n octanoylthioacetyl)piperazine.- The method of Procedure 8 is repeated with substitution an equimolar basis of n-octanoyl chloride for the propionyl chloride specified there.

(15) 1,4 bis(benzoylthioacetyl)piperazine. The method of Procedure 8 is repeated with substitution of an equimolar amount of benzoyl chloride for the propionyl chloride specified in that method; M.P. 188-192" C.; 77% yield.

Analysis.-Calcd. for C H N O S (percent): C, 59.71; H, 5.01; N, 6.33. Found (percent): C, 59.65; H, 5.12, N, 6.41.

(16) 1,4 bis(2 mercaptopentanoyl)piperazine.Two molecular portions of Z-chloropentanoyl chloride are allowed to react with 1 molecular portion piperazine hexahydrate according to the method of Procedure 1. The resulting 1,4-bis(2-chloropentanoyl)piperazine is then converted to the desired product by reaction with potassium thiolacetate according to the method of Procedure 6 to provide 1,4-bis(Z-acetylthiopentanoyl)piperazine, one of the products of the present invention. The latter is then hydrolyzed according to the method of Procedure 7 to give the named product.

(17) Salts.Salts of the products of Procedures 4, 7, 12, and 16 with pharmaceutically acceptable bases are prepared by reaction of chemically equivalent amounts with respect to the SH group of the pharmaceutically acceptable base and the mercaptoacyl compound in a reaction inert solvent such as water, ethanol, or in the judgement of the chemist a relatively non-polar organic solvent such as ether or ethyl acetate. Suitable bases include sodium hydroxide, sodium carbonate, potassium hydroxide, potassium bicarbonate, calcium hydroxide, barium hydroxide, aluminum hydroxide, zinc hydroxide, triethanolamine, triethylamine, diethylamine, tetramethylammonum hydroxide, benzyltrimethyl ammonium hydroxide, etc. The salt is recovered by filtration or evaporation.

What is claimed is:

1. A compound selected from the group consisting of 1,4-bis-acylpiperazines having the formula ,80 ,697. 7 wherein R and R' are hydrogen or lower alkyl hav ing 1 73,347,860 --1( /1 969- ,Frikura 2 60268 C to 4 carbon atoms and X and Y are hydrogen, alkanoyl 3,511,840 1 4-,- 5/1970. -.Tesoro 260-.268 C having up to 20 carbon atoms or benzoyl, and the isalits of 3,'Z26;880 f 1973 Capps 260 .268 C those substances wherein X and Y are hydrogcn with 2,901,481 8/ 1959 Fiisco..- 260v 2 -68 C pharmaceutically acceptable bases. 5 3,660,398 5/1972 Ley 260-268 C .Th dfl' 1h thforul v a 2 e compoun o calm avmg a RE GN ATE Y wherein X and R have the same definitionfa sin cl aim l; m 5

3. The compound of claim 1 wherein Rand'R' are hyh Abstr 6 h Collective X,"P- '9OZ8SYAbStFQCt drogen and X and Y are alkanoyl groups havin'g up tg 2 0 Y 5 1 14940 I h carbon atoms. v Chem. Abstr yol; 68;co1.': 29723c (aJbstractmg Japan 4. 1,4bis(mercaptoacetyl)piperazine. 15 8 I u I 5. l,4-'bis(acetylthioacetyl)piperazine.

References Cited v UNITED STATES PATENTS 2,650,925 9/1653 Ouperoif-urime 260426;; 6 2;; 3,189,590 6/1965 Coover 260 268 c Patent No. 3,809,697 Dated May 7, 1974 InV9nt0r(S) TELLIS' ALEXANDER MARTIN and WILLIQMEIMMEY COMER It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

Column 2, line 47, correct "nucolytic" to read --mucolytic;

"in vitro" is italicized;

line 67, correct "of" to read --on-.

Column 3, line 3, correct "as" second occurrence to read -is-;

line 4, "in vitro" is italicized;

line 61, "in vivo" is italicized. Column 4, line 18, insert a period in place of the coma lines 18, 19 and 20, delete the phrase "namely the substances of Formula I wherein chlorine, bromine, or iodine are substituted for X and Y.";

line 29, delete the word "and".

Column 6, lines 66 and 67, correct "tetramethylanmonum" to read --tetramethy1ammonium--.

Signed and sealed this 8th day of October 1974.

(DEAL) ttest:

McCOY M. GIBSON JR. C. MARSHALL DANN Attesting Officer Commissioner of Patents FGFM PGTQSO (10459) USCOMM-DC wan-Poo W LL51 GOVERNMENT PRINTING OFFICE l9, O-JiE-JSL 

